Tobacco Specific Nitrosamines (TSNAs) are formed primarily during the curing and processing of tobacco leaves. Tobacco curing is a process of physical and biochemical changes that bring out the aroma and flavor of each variety of tobacco. It is believed that the amount TSNA in cured tobacco leaf is dependent on the accumulation of nitrites, which accumulate during the death of the plant cell and are formed during curing by the reduction of nitrates under conditions approaching an anaerobic (oxygen deficient) environment. The reduction of nitrates to nitrites is believed to occur by the action of bacteria on the surface of the leaf under anaerobic conditions, and this reduction is particularly pronounced under certain conditions. Once nitrites are formed, these compounds are believed to combine with various tobacco alkaloids, including pyridine-containing compounds, to form nitrosamines.
The four principal TSNAs, that is, those typically found to be present in the highest concentrations, are N-nitrosonicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). Minor compounds, that is, those typically found at significantly lower levels than the principal TSNAs, include 4-(methylnitrosamino) 4-(3-pyridyl) butanal (NNA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamino)4-(3-pyridyl)-1-butanol (iso-NNAL), and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butyric acid (iso-NNAC). At least NNN and NNK have been reported to be carcinogenic when applied to animals in laboratory studies.
Lowering the concentrations of compounds responsible for the nitrosation of alkaloids to TSNAs can result in decreased TSNA levels in cured leaves. A major nitrosating agent in tobacco leaves is nitrite (NO2−), resulting from the reduction of free nitrate (NO3−) through an enzymatic reaction possibly catalyzed by bacteria during curing. Fertilizer studies altering nitrate levels in Burley plants resulted in different TSNA levels in cured leaves and smoke. Nitrate is the major source of nitrogen available in the soil. In plants, it is absorbed by root epidermal cells and transported to the whole plant to be first reduced to nitrite which is further reduced to ammonia and then assimilated into amino acids. Unfortunately, nitrogen limitation during Burley growth results in unfavorable agronomic phenotypes such as poor biomass yield and delay in plant maturation and is therefore not a commercially viable approach to reduce TSNA levels. Trying to manipulate nitrate accumulation in tobacco leaf is a major challenge.
WO98/58555 describes the treatment of tobacco leaves before or during flue-curing by microwaving for reducing TSNAs. U.S. Pat. No. 5,810,020 describes a process for removing TSNAs from tobacco by contacting the tobacco material with a trapping sink, wherein the trapping sink comprises a select transition metal complex which is readily nitrosated to form a nitrosyl complex with little kinetic or thermodynamic hindrance. U.S. Pat. No. 6,202,649 describes a method of substantially preventing formation of TSNAs by, among other things, curing tobacco in a controlled environment having a sufficient airflow to substantially prevent an anaerobic condition around the vicinity of the tobacco leaf. The controlled environment is provided by controlling one or more curing parameters, such as airflow, humidity, and temperature. However, methods such as these can add considerable cost and time to the production of tobacco and therefore are less likely to be accepted by the tobacco industry. Thus, a need remains for an effective and relatively inexpensive method for reducing TSNAs.
Molecular based methods for reducing the levels of TSNAs in plants are highly desirable since they do not require expensive, and often complex, methods to achieve the reduced levels of TSNAs. One such molecular based approach is disclosed in WO2011/088180. Compositions and methods are disclosed for inhibiting the expression or function of root-specific nicotine demethylase polypeptides that are involved in the metabolic conversion of nicotine to nornicotine in the roots of tobacco plants. The gene sequence of the CYP82E10 nicotine demethylase gene is disclosed. Reducing the expression of this gene was found to reduce the levels of NNN in cured tobacco leaves. Whilst reduced levels of NNN may be obtained, there is more than one TSNA that has been reported to be carcinogenic which will still remain in the modified plants. Other nicotine demethylase genes include CYP82E4 and CYP82E5 which participate in the conversion of nicotine to nornicotine and are described in WO2006091194, WO2008070274 and WO2009064771.
There is a continuing need in the art to develop molecular based strategies for reducing the levels of TSNAs in cured tobacco leaves. The present invention seeks to address this need.